Abstract

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 µm away.

© 2005 Optical Society of America

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Angiogenesis. (1)

Y. Numaguchi, S. Huang, T.R. Polte, G.S. Eichler, N. Wang, and D.E. Ingber, �??Caldesmon-dependent switching between capillary endothelial cell growth and apoptosis through modulation of cell shape and contractility,�?? Angiogenesis. 6, 55-64 (2003)
[CrossRef] [PubMed]

Appl. Phys. A (1)

C.B. Schaffer, J.F. Garcia, and E. Mazur, �??Bulk heating of transparent materials using a high-repetition-rate femtosecond laser,�?? Appl. Phys. A 76, 351-354 (2003)
[CrossRef]

Arch. Ophthalmol. (1)

D. Stern, R.W. Schoenlein, C.A. Puliafito, E.T. Dobi, R. Birngruber, and J.G. Fujimoto, �??Ablation by Nanosecond, Picosecond, and Femtosecond Lasers at 532 nm and 625 nm,�?? Arch. Ophthalmol. 107, 587-592 (1989)
[CrossRef] [PubMed]

Biophys. (1)

E.L.Botvinick, V.Venugopalan, J.V.Shah, L.H.Liaw, and M.W. Berns, �??Controlled Ablation of Microtubules Using a Picosecond Laser,�?? Biophys. J. 87, 6, 4203-4212 (2004)
[CrossRef] [PubMed]

Cell. Mol. Biol. (1)

K. Koenig, I. Riemann, P. Fischer, and K. Halbhuber, �??Intracellular Nanosurgery With Near Infrared Femtosecond Laser Pulses,�?? Cell. Mol. Biol. 45, 192-201 (1999)

Chem. Rev. (1)

A. Vogel, and V. Venugopalan, �??Mechanisms of Pulsed Laser Ablation of Biological Tissues,�?? Chem. Rev. 103, 2, 577-644 (2003)
[CrossRef] [PubMed]

CLEO 2001 (1)

N. Shen, C.B. Schaffer, D. Datta, and E. Mazur, �??Photodisruption in biological tissues and single cells using femtosecond laser pulses,�?? in Lasers and Electro Optics, conference technical digest, OSA, Washington, DC, 56, 403-404 (2001)

Exp. Cell Res. (1)

F.J. Alenghat, S.M. Nauli, R. Kolb, J. Zhou, and D.E. Ingber, �??Global cytoskeletal control of mechanotransduction in kidney epithelial cells,�?? Exp. Cell Res. 301, 23-30 (2004)
[CrossRef] [PubMed]

Experimental Cell Research (1)

H. Liang, W.H. Wright, S. Cheng, W. He, and M.W. Berns, �??Micromanipulation of Chromosomes in PTK2 Cells Using Laser Microsurgery (Optical Scalpel) in Combination with Laser-Induced Optical Force (Optical Tweezers), �?? Experimental Cell Research 204, 110-120 (1993)
[CrossRef] [PubMed]

Front. Biosci. (1)

S.H. Hu, J.X. Chen, and N. Wang, �??Cell spreading controls balance of prestress by microtubules and extracellular matrix,�?? Front. Biosci. 9, 2177-2182 (2004)
[CrossRef] [PubMed]

J. Appl. Phys. (1)

J. Noack, D.X. Hammer, G.D. Noojin, B.A. Rockwell, and A. Vogel, �??Influence of pulse duration on mechanical effects after laser-induced breakdown in water,�?? J. Appl. Phys. 83, 12, 7488-7495 (1998)
[CrossRef]

J. Microscopy (3)

K. Koenig, �??Multiphoton Microscopy in Life Sciences,�?? J. Microscopy 200, 83-104 (2000)
[CrossRef]

D. Gusnard, and R. H. Kirschner, �??Cell and organelle shrinkage during preparation for scanning electron microscopy: Effects of fixation, dehydration and critical point drying,�?? J. Microscopy 110, 1, 51-57 (1977)
[CrossRef]

U. Brunk, V.P. Collins, and E. Arro, �??The fixation, dehydration, drying and coating of cultured cells of SEM,�?? J. Microscopy 123, 2, 121-131 (1981)
[CrossRef]

Journal of Cell Science (1)

J.R. Aist, H. Liang, and M.W. Berns, �??Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study,�?? Journal of Cell Science 104, 1207-1216 (1993)
[PubMed]

Nature (2)

M.F. Yanik, H. Cinar, H.N. Cinar, A.D. Chisholm, Y. Jin, and A. Ben-Yakar, �??Neurosurgery: Functional regeneration after laser axotomy,�?? Nature 432, 822 (2004)
[CrossRef] [PubMed]

U.K. Tirlapur, and K. Koenig, �??Targeted transfection by femtosecond laser,�?? Nature 448, 290-291 (2002)
[CrossRef]

Opt. Express (2)

Phys. Rev. Lett. (1)

V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, �??Role of Laser-Induced Plasma Formation in Pulsed Cellular Microsurgery and Micromanipulation,�?? Phys. Rev. Lett. 88, 078103, 1-4 (2002)
[CrossRef]

Science (4)

W. Denk, J.H. Strickler, and W.W. Webb, �??Two-Photon Laser Scanning Fluorescence Microscopy,�?? Science 248, 4951, 73-76 (1990)
[CrossRef] [PubMed]

C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides, and D.E. Ingber, �??Geometric Control of Cell Life and Death,�?? Science 276, 1425-1427 (1997)
[CrossRef] [PubMed]

S.W. Grill, J. Howard, E. Schäffer, E.H.K. Stelzer, and A.A. Hyman, �??The Distribution of Active Force Generators Controls Mitotic Spindle Position,�?? Science 301, 518-521 (2003)
[CrossRef] [PubMed]

M.W. Berns, J. Aist, J. Edwards, K. Strahs, J. Girton, P. McNeil, J.B. Rattner, M. Kitzes, M. Hammerwilson, L.H. Liaw, A. Siemens, M. Koonce, S. Peterson, S. Brenner, J. Burt, R. Walter, P. J. Bryant, D. Vandyk, J. Couclombe, T. Cahill, and G.S. Bern, �??Laser microsurgery in cell and developmental biology,�?? Science 213, 505-513 (1981)
[CrossRef] [PubMed]

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Figures (4)

Fig. 1.
Fig. 1.

(a) Cuts through fluorescently-labeled actin fibers in a fixed endothelial cell obtained by irradiation with femtosecond laser pulses of energies between 1.8 nJ and 4.4 nJ. (b) Fluorescence intensity profile along the actin bundle outlined in the image

Fig. 2.
Fig. 2.

Cuts in the nucleus of a fixed endothelial cell at various laser energies, imaged by (a) fluorescence microscopy and (b) electron microscopy.

Fig. 3.
Fig. 3.

Pulse energy dependence of the ablation width of cuts in the nucleus of endothelial cells measured by fluorescence microscopy (filled circles) and TEM (open circles) in three different cells (a)–(c).

Fig. 4.
Fig. 4.

(a) Fluorescence microscope image of GFP-labeled microtubule network in an endothelial cell. (b) time-lapse sequence showing rapid retraction of microtubule due to depolymerization. The cross hair shows the position targeted by the laser; the triangles show the retracting ends of the microtubule.

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